Cloning, Expression And Founction Analysis Of Chitinase Genes From Wheat Xinong 538

Chitinase, which is an important defensive factor in plants, is closely related to plant disease resistance. In order to expand the chitinase gene resources, understand the expression and fuction of chitinase genes in wheat. By the using of universal primer ChiF/ChiR, chitinase genes were cloned by PCR from 10 wheat cultivars with different resistance to disease in this study. After that, these sequences were analyzed by different biology software, and were expressed in E.coli. The expression of these genes in Xinong538’s root、stem and leaf were analyzed by Quantitative Real-time PCR. The higher amounts of two chitinase genes in these organization were selected to test the resistance with Stripe rust infection. The results showed that:(1) 5 chitinase genes (960±3 bp in length) were cloned from 10 wheat cultivars with different resistance to disease, named Chtl, Cht2, Cht3, Cht4 and Cht5 in turn. By the using of BLAST online software to analysis the homology comparison with these five gene sequences in NCBI,100 nucleotide sequences were found in homologous, Wherein Cht5 gene is fully consistent with Chi-3 gene (accession number is KJ507387) from Chinese Spring, then submitted the others to GenBank, accession number was:KR049247-KR049250 in turn.(2) To analysis the new sequences, the result showed that these chitinase genes in this research had a complete open reading frame without introns, the initiation codon was ATG, termination codon was TAG The similarity of nucleotide and amino acid sequences among Chtl-Cht4 was 96.84% and 91.02% respectively. The encoding chitinase based on these genes were hydrophilic proteins, besides that they belonged to Class I b type, and they were the members of glycoside hydrolase family 19, they were extracellular secreted protein and had lysozyme activity at the same time. The known sources of different types of chitinase protein sequence were obtained by NCBI to alignment and struct phylogenetic trees with the four amino acid sequences, clustering results showed that chitinase in this research belonging to Class I type,too.(3) The results of SDS-PAGE showed that recombinant plasmid could express a 33KD protein as inclusion body after induction in TransBL21(DE3),which wass consistent with the size of the target protein.(4) The results of quantitative PCR showed that these 5 kinds of chitinase genes were expressed in Xinong538’s roots, stems and leaves, however, the amount of expression in different tissues are quite different. The content of any chitinase genes in this experiment was highest in leaf, followed by root, and is minimum in stem; in the mean time, the expression of these five kinds chitinase genes in the same organization were: Cht4>Cht2>Cht3>Cht1>Chi-3. In this study, the chitinase genes were constitutively expressed in wheat, indicating their participation in the regular growth and development of wheat.(5)Therefore, Cht4 and Cht2, which had higher content in the same organization, were selected to validate the ability of their resistance to fungal diseases. The results showed that the expression of the two genes can be induced by Puccinia striiformis f.sp.tritici, and the expression peak time of Cht4 was earlier than Cht2 genes. The expression of Cht4 in 92R137 (resistant material) was significantly higher than the Xinong 538(moderately resistant material) and Mingxian 169 (susceptible material), and all of them were higher than the expression in leaves, which were inoculated with ddH2O in the same conditions. The expression of Cht2 in Xinong 538(moderately resistant material) was significantly higher than the 92R137 (resistant material) and Mingxian 169 (susceptible material), the expression levels of Cht2 were higher than controls in resistant material, but almost had the same level with control in Mingxian 169(susceptible material). The results showed that under Puccinia striiformis f.sp.tritici stress, the expression of Cht4 gene and Cht2 gene were induced by stripe rust, suggesting that they may be involved in the defense reaction for Puccinia striiformis f.sp.tritici in wheat leaves.